The mir-7 and bag of marbles genes regulate Hedgehog pathway signaling in blood cell progenitors in Drosophila larval lymph glands.

Hedgehog (Hh) pathway signaling is crucial for the maintenance of blood cell progenitors in the lymph gland hematopoietic organ present in Drosophila third instar larvae. Previous studies from our lab have likewise shown the importance of the mir-7 and bag of marbles (bam) genes in maintaining the progenitor state. Thus, we sought to investigate a possible interaction between the Hh pathway and mir-7/bam in the prohemocyte population within this hematopoietic tissue. Gain of function mir-7 was able to rescue a blood cell progenitor depletion phenotype caused by Patched (Ptc) inhibition of Hh pathway signaling in these cells. Similarly, expression of a dominant/negative version of Ptc was able to rescue the severe reduction of prohemocytes due to bam loss of function. Furthermore, we demonstrated that Suppressor of fused [Su(fu)], another known inhibitor of Hh signaling, likely serves as a translational repression target of the mir-7 miRNA. Our results suggest the mir-7/bam combination regulates the Hh signaling network through repression of Su(fu) to maintain hemocyte progenitors in the larval lymph gland.

Differential requirement of SUFU in tissue development discovered in a hypomorphic mouse model.

Suppressor of Fused (SUFU) is an essential negative regulator of the Hedgehog (HH) pathway and involved in GLI transcription factor regulation. Due to early embryonic lethality of Sufu-/- mice, investigations of SUFU's role later in development are limited to conditional, tissue-specific knockout models. In this study we developed a mouse model (SufuEx456(fl)/Ex456(fl)) with hypomorphic features where embryos were viable up to E18.5, although with a spectrum of developmental defects of varying severity, including polydactyly, exencephaly and omphalocele. Development of certain tissues, like the skeleton, was more affected than that of others such as skin, which remained largely normal. Interestingly, no apparent changes in the dorso-ventral patterning of the neural tube at E9.0 could be seen. Thus, this model provides an opportunity to globally study SUFU's molecular function in organogenesis beyond E9.5. Molecularly, SufuEx456(fl)/Ex456(fl) embryos displayed aberrant mRNA splicing and drastically reduced levels of Sufu wild-type mRNA and SUFU protein in all tissues. As a consequence, at E9.5 the levels of all three different GLI proteins were reduced. Interestingly, despite the reduction of GLI3 protein levels, the critical ratio of the GLI3 full-length transcriptional activator versus GLI3 truncated repressor remained unchanged compared to wild-type embryos. This suggests that the limited amount of SUFU protein present is sufficient for GLI processing but not for stabilization. Our data demonstrate that tissue development is differentially affected in response to the reduced SUFU levels, providing novel insight regarding the requirements of different levels of SUFU for proper organogenesis.

miR-423-5p inhibits myoblast proliferation and differentiation by targeting Sufu.

Myoblast proliferation and terminal differentiation are the key steps of myogenesis. MicroRNAs are a class of small noncoding RNAs that play important roles in gene expression regulation. They negatively regulate gene expression by causing messenger RNA translational repression or target messenger RNA degradation. Here, we found that microRNA-423-5p (miR-423-5p) is highly expressed in both slow and fast muscles. Our gain-of-function study indicated that miR-423-5p actually plays a negative role in regulating myoblast proliferation and differentiation. We also found that miR-423-5p is able to inhibit the expression of suppressor of fused homolog to inactivate the expression of the marker genes in myoblast proliferation and differentiation. Taken together, our findings indicated miR-423-5p as a potential inhibitor of myogenesis by targeting suppressor of fused homolog in myoblast, and it also contributes to a better understanding of the microRNAs-target gene regulatory network in different types of porcine muscle types and may benefit the practice of improving the meat quality in animal husbandry.

Development of thieno- and benzopyrimidinone inhibitors of the Hedgehog signaling pathway reveals PDE4-dependent and PDE4-independent mechanisms of action.

From a high content in vivo screen for modulators of developmental patterning in embryonic zebrafish, we previously identified eggmanone (EGM1, 3) as a Hedgehog (Hh) signaling inhibitor functioning downstream of Smoothened. Phenotypic optimization studies for in vitro probe development utilizing a Gli transcription-linked stable luciferase reporter cell line identified EGM1 analogs with improved potency and aqueous solubility. Mechanistic profiling of optimized analogs indicated two distinct scaffold clusters: PDE4 inhibitors able to inhibit downstream of Sufu, and PDE4-independent Hh inhibitors functioning between Smo and Sufu. Each class represents valuable in vitro probes for elucidating the complex mechanisms of Hh regulation.

Active signals, gradient formation and regional specificity in neural induction.

The question of how the vertebrate embryo gives rise to a nervous system is of paramount interest in developmental biology. Neural induction constitutes the earliest step in this process and is tightly connected with development of the embryonic body axes. In the Xenopus embryo, perpendicular gradients of BMP and Wnt signals pattern the dorsoventral and anteroposterior body axes. Both pathways need to be inhibited to allow anterior neural induction to occur. FGF8 and IGF are active neural inducers that together with BMP and Wnt signals are integrated at the level of Smad 1/5/8 phosphorylation. Hedgehog (Hh) also contributes to anterior neural induction. Suppressor-of-fused plays an important role in intertwining the Hh and Wnt pathways. Distinct mechanisms are discussed that establish morphogen gradients and integrate retinoic acid and FGF signals during posterior development. These findings not only improve our understanding of regional specification in neural induction, but have profound implications for mammalian stem cell research and regenerative medicine.

WNT5A, ß­catenin and SUFU expression patterns, and the significance of microRNA deregulation in placentas with intrauterine growth restriction.

Placental insufficiency is a common cause of intrauterine growth restriction (IUGR). It affects ~10% of pregnancies and increases fetal and neonatal morbidity and mortality. Although Wnt and Hh pathways are crucial for embryonic development and placentation, their role in the pathology of IUGR is still not sufficiently explored. The present study analyzed the expression of positive regulators of the Wnt pathway, WNT5A and ß­catenin, and the expression of the Hh pathway negative regulator suppressor of fused (SUFU). Immunohistochemical and reverse transcription­quantitative PCR (RT­qPCR) assays were performed on 34 IUGR and 18 placental tissue samples from physiologic singleton­term pregnancies. Epigenetic mechanisms of SUFU gene regulation were also investigated by methylation­specific PCR analysis of its promoter and RT­qPCR analysis of miR­214­3p and miR­378a­5p expression. WNT5A protein expression was higher in endothelial cells of placental villi from IUGR compared with control tissues. That was also the case for ß­catenin protein expression in trophoblasts and endothelial cells and SUFU protein expression in trophoblasts from IUGR placentas. The SUFU gene promoter remained unmethylated in all tissue samples, while miR­214­3p and miR­378a­5p were downregulated in IUGR. The present results suggested altered Wnt and Hh signaling in IUGR. DNA methylation did not appear to be a mechanism of SUFU regulation in the pathogenesis of IUGR, but its expression could be regulated by miRNA targeting.

Suppressor of Fused Regulation of Hedgehog Signaling is Required for Proper Astrocyte Differentiation.

Hedgehog signaling is essential for vertebrate development; however, less is known about the negative regulators that influence this pathway. Using the mouse P19 embryonal carcinoma cell model, suppressor of fused (SUFU), a negative regulator of the Hedgehog (Hh) pathway, was investigated during retinoic acid (RA)-induced neural differentiation. We found Hh signaling increased activity in the early phase of differentiation, but was reduced during terminal differentiation of neurons and astrocytes. This early increase in pathway activity was required for neural differentiation; however, it alone was not sufficient to induce neural lineages. SUFU, which regulates signaling at the level of Gli, remained relatively unchanged during differentiation, but its loss through CRISPR-Cas9 gene editing resulted in ectopic expression of Hh target genes. Interestingly, these SUFU-deficient cells were unable to differentiate toward neural lineages without RA, and when directed toward these lineages, they showed delayed and decreased astrocyte differentiation; neuron differentiation was unaffected. Ectopic activation of Hh target genes in SUFU-deficient cells remained throughout RA-induced differentiation and this was accompanied by the loss of Gli3, despite the presence of the Gli3 message. Thus, the study indicates the proper timing and proportion of astrocyte differentiation requires SUFU, likely acting through Gli3, to reduce Hh signaling during late-stage differentiation.

Suppressor of fused-restrained Hedgehog signaling in chondrocytes is critical for epiphyseal growth plate maintenance and limb elongation in juvenile mice.

Hedgehog (Hh) signaling plays multiple critical roles in regulating chondrocyte proliferation and differentiation during epiphyseal cartilage development. However, it is still unclear whether Hh signaling in chondrocytes is required for growth plate maintenance during juvenile growth, and whether sustained activation of Hh signaling in chondrocytes promotes limb elongation. In this study, we first utilized Hh reporter mice to reveal that Hh signaling was activated in resting and columnar chondrocytes in growth plates of juvenile and adult mice. Next, we genetically modulated Hh signaling by conditionally deleting Smo or Sufu in all or a subpopulation of growth plate chondrocytes, and found that ablation of either Smo or Sufu in chondrocytes of juvenile mice caused premature closure of growth plates and shorter limbs, whereas Osx-Cre-mediated deletion of either of these two genes in prehypertrophic chondrocytes did not lead to obvious growth plate defects, indicating that Hh signaling mainly functions in resting and/or columnar chondrocytes to maintain growth plates at the juvenile stage. At the cellular level, we found that chondrocyte-specific ablation of Smo or Sufu accelerated or suppressed chondrocyte hypertrophy, respectively, whereas both decreased chondrocyte proliferation and survival. Thus, our study provided the first genetic evidence to establish the essential cell-autonomous roles for tightly-regulated Hh signaling in epiphyseal growth plate maintenance and limb elongation during juvenile growth.

Suppressor of fused associates with dissemination patterns in patients with glioma.

Gliomas are the most common brain tumors, which present poor prognosis, due, in part, to tumor cell migration and infiltration into distant brain areas. However, the underlying mechanisms causing such effects are unknown. Hedgehog (HH)-Gli axis is one of the signaling pathways involved, with a high number of molecular mediators. In this study, we investigated the association between HH-Gli intermediates and clinical parameters. We found that high levels of SuFu are associated with high dissemination patterns in patients with glioma. Therefore, we analyzed SuFu expression data in three glioma cohorts of surgical samples (N =1,759) and modified its expression in Glioblastoma Cancer Stem Cells (GB CSC) in vitro models. Our data reveal that SuFu overexpression increases cancer stemness properties together with a migratory phenotype. This work identifies SuFu as a new molecular player in glioma cell migration and a promising target to develop blocking agents to decrease GB dissemination.

Hedgehog signaling in gastrointestinal carcinogenesis and the gastrointestinal tumor microenvironment.

The Hedgehog (HH) signaling pathway plays important roles in gastrointestinal carcinogenesis and the gastrointestinal tumor microenvironment (TME). Aberrant HH signaling activation may accelerate the growth of gastrointestinal tumors and lead to tumor immune tolerance and drug resistance. The interaction between HH signaling and the TME is intimately involved in these processes, for example, tumor growth, tumor immune tolerance, inflammation, and drug resistance. Evidence indicates that inflammatory factors in the TME, such as interleukin 6 (IL-6) and interferon-γ (IFN-γ), macrophages, and T cell-dependent immune responses, play a vital role in tumor growth by affecting the HH signaling pathway. Moreover, inhibition of proliferating cancer-associated fibroblasts (CAFs) and inflammatory factors can normalize the TME by suppressing HH signaling. Furthermore, aberrant HH signaling activation is favorable to both the proliferation of cancer stem cells (CSCs) and the drug resistance of gastrointestinal tumors. This review discusses the current understanding of the role and mechanism of aberrant HH signaling activation in gastrointestinal carcinogenesis, the gastrointestinal TME, tumor immune tolerance and drug resistance and highlights the underlying therapeutic opportunities.

Suppressor of Fused Inhibits Skin Wound Healing.

Objectives: To investigate the effect of suppressor of fused (Sufu) on epidermal and dermal cellular properties and in wound healing. Approach: Transgenic (TG) mice overexpressing human Sufu (hSufu) in the epidermis were applied to investigate the effects of Sufu on epidermal and dermal cellular properties and in wound healing. Results: Histological staining revealed a reduction of epidermal and dermal thickness and an increase of hypodermal adipose tissue in homozygous K14-hSufu TG mice when compared with wild-type (WT) controls. TG mice exhibited significantly delayed skin wound healing. Moreover, the migratory and proliferative capabilities of cultured keratinocytes were decreased in K14-hSufuTG mice. Transforming growth factor-ß treatment increased the expression of α-smooth muscle actin more in WT than in TG fibroblasts. Sufu overexpression significantly decreased the expression of ß-catenin, glioma transcription factor 1 (Gli1), and matrix metalloproteinase-3 in wounds of K14-hSufu TG mice when compared with controls, probably indicating a delaying effect of Sufu on wound healing via blocking the hedgehog (Hh)/Gli and Wnt/ß-catenin pathway. Innovation: Our results indicate a new property of Sufu in the process of skin wound healing. It provides an important basis for Sufu as a potential target for skin wound healing. Conclusion: Our findings suggest that Sufu overexpression in the epidermis impairs wound healing via dampening the Hh/Gli and Wnt/ß-catenin signaling pathway. These data provide an important basis for further analyses of Sufu in skin wound healing.

miR-214-3p-Sufu-GLI1 is a novel regulatory axis controlling inflammatory smooth muscle cell differentiation from stem cells and neointimal hyperplasia.

BACKGROUND: Inflammatory smooth muscle cells (iSMCs) generated from adventitial stem/progenitor cells (AdSPCs) have been recognised as a new player in cardiovascular disease, and microRNA-214-3p (miR-214-3p) has been implicated in mature vascular SMC functions and neointimal hyperplasia. Here, we attempted to elucidate the functional involvements of miR-214-3p in iSMC differentiation from AdSPCs and unravel the therapeutic potential of miR-214-3p signalling in AdSPCs for injury-induced neointimal hyperplasia. METHODS: The role of miR-214-3p in iSMC differentiation from AdSPCs was evaluated by multiple biochemistry assays. The target of miR-214-3p was identified through binding site mutation and reporter activity analysis. A murine model of injury-induced arterial remodelling and stem cell transplantation was conducted to study the therapeutic potential of miR-214-3p. RT-qPCR analysis was performed to examine the gene expression in healthy and diseased human arteries. RESULTS: miR-214-3p prevented iSMC differentiation/generation from AdSPCs by restoring sonic hedgehog-glioma-associated oncogene 1 (Shh-GLI1) signalling. Suppressor of fused (Sufu) was identified as a functional target of miR-214-3p during iSMC generation from AdSPCs. Mechanistic studies revealed that miR-214-3p over-expression or Sufu inhibition can promote nuclear accumulation of GLI1 protein in AdSPCs, and the consensus sequence (GACCACCCA) for GLI1 binding within smooth muscle alpha-actin (SMαA) and serum response factor (SRF) gene promoters is required for their respective regulation by miR-214-3p and Sufu. Additionally, Sufu upregulates multiple inflammatory gene expression (IFNγ, IL-6, MCP-1 and S100A4) in iSMCs. In vivo, transfection of miR-214-3p into the injured vessels resulted in the decreased expression level of Sufu, reduced iSMC generation and inhibited neointimal hyperplasia. Importantly, perivascular transplantation of AdSPCs increased neointimal hyperplasia, whereas transplantation of AdSPCs over-expressing miR-214-3p prevented this. Finally, decreased expression of miR-214-3p but increased expression of Sufu was observed in diseased human arteries. CONCLUSIONS: We present a previously unexplored role for miR-214-3p in iSMC differentiation and neointima iSMC hyperplasia and provide new insights into the therapeutic effects of miR-214-3p in vascular disease.

Sulforaphane inhibits the activation of hepatic stellate cell by miRNA-423-5p targeting suppressor of fused.

Liver fibrosis, a common pathological process in chronic liver diseases, is characterized by excessive accumulation of extracellular matrix proteins and considered as a wound healing response to chronic liver injury. Hepatic stellate cell (HSC) activation plays a key role in liver fibrosis development. Previous studies showed that sulforaphane (SFN) has wide protective effects against tissue injury and inflammation. Accumulating evidence has shown that microRNAs play important roles in the development of hepatic fibrosis, some of which have been identified as potential therapeutic targets. This study was conducted to explore the role of SFN in the suppression of HSC activation. Quantitative real-time PCR showed that HSC miR-423-5p levels were up-regulated during HSC activation and down-regulated after SFN administration. Further, transfection of a miR-423-5p mimic demonstrated that inhibition of HSC activation by SFN required down-regulation of miR-423-5p. We showed that suppressor of fused is the direct target of miR-423-5p. SFN may play a role in inhibiting hepatic fibrosis by downregulating miRNA-423-5p. MiRNA-423-5p may be useful as a therapeutic target for treating hepatic fibrosis.

Molecular mechanisms of suppressor of fused in regulating the hedgehog signalling pathway.

Highly conserved throughout evolution, the hedgehog (Hh) signalling pathway has been demonstrated to be involved in embryonic development, stem cell maintenance and tissue homeostasis in animals ranging from invertebrates to vertebrates. In the human body, a variety of cancer types are associated with the aberrantly activated Hh signalling pathway. Multiple studies have revealed suppressor of fused (Sufu) as a key negative regulator of this signalling pathway. In vertebrates, Sufu primarily functions as a tumor suppressor factor by interacting with and inhibiting glioma-associated oncogene homologues (GLIs), which are the terminal transcription factors of the Hh signalling pathway and belong to the Kruppel family of zinc finger proteins; by contrast, the regulation of Sufu itself remains relatively unclear. In the present review article, we focus on the effects of Sufu on the Hh signalling pathway in tumourigenesis and the molecular mechanisms underlying the regulation of GLI by Sufu. In addition, the factors modulating the activity of Sufu at post-transcriptional levels are also discussed.

A familial syndrome of hypothalamic hamartomas, polydactyly, and SMO mutations: a clinical report of 2 cases.

Hypothalamic hamartomas are benign tumors known to cause gelastic or dacrystic seizures, precocious puberty, developmental delay, and medically refractory epilepsy. These tumors are most often sporadic but rarely can be associated with Pallister-Hall syndrome, an autosomal dominant familial syndrome caused by truncation of glioblastoma transcription factor 3, a downstream effector in the sonic hedgehog pathway. In this clinical report, the authors describe two brothers with a different familial syndrome. To the best of the authors' knowledge, this is the first report in the literature describing a familial syndrome caused by germline mutations in the Smoothened (SMO) gene and the first familial syndrome associated with hypothalamic hamartomas other than Pallister-Hall syndrome. The authors discuss the endoscopic endonasal biopsy and subtotal resection of a large hypothalamic hamartoma in one of the patients as well as the histopathological findings encountered. Integral to this discussion is the understanding of the hedgehog pathway; therefore, the underpinnings of this pathway and its clinical associations to date are also reviewed.

Suppressor of Fused Chaperones Gli Proteins To Generate Transcriptional Responses to Sonic Hedgehog Signaling.

Cellular responses to the graded Sonic Hedgehog (Shh) morphogenic signal are orchestrated by three Gli genes that give rise to both transcription activators and repressors. An essential downstream regulator of the pathway, encoded by the tumor suppressor gene Suppressor of fused (Sufu), plays critical roles in the production, trafficking, and function of Gli proteins, but the mechanism remains controversial. Here, we show that Sufu is upregulated in active Shh responding tissues and accompanies Gli activators translocating into and Gli repressors out of the nucleus. Trafficking of Sufu to the primary cilium, potentiated by Gli activators but not repressors, was found to be coupled to its nuclear import. We have identified a nuclear export signal (NES) motif of Sufu in juxtaposition to the protein kinase A (PKA) and glycogen synthase kinase 3 (GSK3) dual phosphorylation sites and show that Sufu binds the chromatin with both Gli1 and Gli3. Close comparison of neural tube development among individual Ptch1-/-, Sufu-/-, and Ptch1-/-; Sufu-/- double mutant embryos indicates that Sufu is critical for the maximal activation of Shh signaling essential to the specification of the most-ventral neurons. These data define Sufu as a novel class of molecular chaperone required for every aspect of Gli regulation and function.

Cadherin-7 enhances Sonic Hedgehog signalling by preventing Gli3 repressor formation during neural tube patterning.

Sonic Hedgehog (Shh) is a ventrally enriched morphogen controlling dorsoventral patterning of the neural tube. In the dorsal spinal cord, Gli3 protein bound to suppressor-of-fused (Sufu) is converted into Gli3 repressor (Gli3R), which inhibits Shh-target genes. Activation of Shh signalling prevents Gli3R formation, promoting neural tube ventralization. We show that cadherin-7 (Cdh7) expression in the intermediate spinal cord region is required to delimit the boundary between the ventral and the dorsal spinal cord. We demonstrate that Cdh7 functions as a receptor for Shh and enhances Shh signalling. Binding of Shh to Cdh7 promotes its aggregation on the cell membrane and association of Cdh7 with Gli3 and Sufu. These interactions prevent Gli3R formation and cause Gli3 protein degradation. We propose that Shh can act through Cdh7 to limit intracellular movement of Gli3 protein and production of Gli3R, thus eliciting more efficient activation of Gli-dependent signalling.

Loss of Suppressor of Fused in Mid-Corticogenesis Leads to the Expansion of Intermediate Progenitors.

Neural progenitors in the embryonic neocortex must be tightly regulated in order to generate the correct number and projection neuron subtypes necessary for the formation of functional neocortical circuits. In this study, we show that the intracellular protein Suppressor of Fused (Sufu) regulates the proliferation of intermediate progenitor (IP) cells at later stages of corticogenesis to affect the number of Cux1+ upper layer neurons in the postnatal neocortex. This correlates with abnormal levels of the repressor form of Gli3 (Gli3R) and the ectopic expression of Patched 1 (Ptch1), a Sonic Hedgehog (Shh) target gene. These studies reveal that the canonical role of Sufu as an inhibitor of Shh signaling is conserved at later stages of corticogenesis and that Sufu plays a crucial role in regulating neuronal number by controlling the cell cycle dynamics of IP cells in the embryonic neocortex.

Nek2A phosphorylates and stabilizes SuFu: A new strategy of Gli2/Hedgehog signaling regulatory mechanism.

Suppressor of Fused (SuFu) plays a conservative role in the regulation of the Gli transcription factors within the Hedgehog (Hh) signaling pathway. Despite the central importance of SuFu in the Hh pathway, little is known about its regulation. Here, we performed a GAL4-based yeast two-hybrid screen using human SuFu as bait, and identified NIMA-related expressed kinase 2A (Nek2A) as a new SuFu-interacting protein, which was also confirmed by glutathione-S-transferase pull-down and co-immunoprecipitation assays. Intriguingly, Nek2A is found to stabilize SuFu at least partly depending on its kinase activity, thereby triggering phosphorylation of the SuFu protein. Moreover, the phosphorylated SuFu inhibits the nuclear localization and transcriptional activity of Gli2/Hh signaling. These findings reveal a new mechanism of mammalian SuFu regulation, and offers novel insights into Hh signaling regulation in development and human disease.